The overall objectives of this project are to isolate and characterize various components of epithelial basement membrane (EBM) associated with both normal and neoplastic cells. (1) Components of EBM synthesized by neoplastic cells will be obtained from murine parietal yolk sac carcinoma (PYS) cells grown in vitro. A glycoprotein which has been isolated from this system in a soluble form has been partially purified and characterized as to amino acid and sugar composition. Further studies will include disaggregation by the use of chemical denaturants, reducing agents and detergents in order to determine the molecular size and biochemical composition of subunits; the examination of various protein fractions obtained during the isolation of the above glycoprotein for the presence of other glycoprotein components of EBM: the mechanism of heterosaccharide elongation during the synthesis of EBM glycoprotein in vitro will be studied in a modified system which results in the secretion of a glycoprotein virtually depleted of sugar; and the isolation and characterization of the collagen component of EBM synthesized by PYS cells. (2) Components of EBM synthesized by murine tissues, primarily the kidney or lens, will be isolated in a soluble form using methods successful for the PYS system. Biochemical and physical characteristics of the isolated components will be determined and compared with those of EBM synthesized by neoplastic cells. (3) Since the EBM in mammalian tissues exists as a complex consisting of both collagen and glycoprotein, the isolated components will be recombined in order to determine the mechanism of complex formation and subsequent deposition of EBM in the extracellular space. BIBLIOGRAPHIC REFERENCE: Johnson, Lewis D., Isolation of a soluble epithelial basement membrane glycoprotein from murine kidney, Fed. Proc. 34: 821 (Abst. #3397), 1975.